www.nature.com/scientificreports OPEN Fungal endophytes from arid areas of Andalusia: high potential sources for antifungal and antitumoral Received: 2 January 2018 Accepted: 19 June 2018 agents Published: xx xx xxxx Victor González-Menéndez1, Gloria Crespo1, Nuria de Pedro1, Caridad Diaz1, Jesús Martín1, Rachel Serrano1, Thomas A. Mackenzie1, Carlos Justicia1, M. Reyes González-Tejero2, M. Casares2, Francisca Vicente1, Fernando Reyes 1, José R. Tormo1 & Olga Genilloud1 Native plant communities from arid areas present distinctive characteristics to survive in extreme conditions. The large number of poorly studied endemic plants represents a unique potential source for the discovery of novel fungal symbionts as well as host-specifc endophytes not yet described. The addition of adsorptive polymeric resins in fungal fermentations has been seen to promote the production of new secondary metabolites and is a tool used consistently to generate new compounds with potential biological activities. A total of 349 fungal strains isolated from 63 selected plant species from arid ecosystems located in the southeast of the Iberian Peninsula, were characterized morphologically as well as based on their ITS/28S ribosomal gene sequences. The fungal community isolated was distributed among 19 orders including Basidiomycetes and Ascomycetes, being Pleosporales the most abundant order. In total, 107 diferent genera were identifed being Neocamarosporium the genus most frequently isolated from these plants, followed by Preussia and Alternaria. Strains were grown in four diferent media in presence and absence of selected resins to promote chemical diversity generation of new secondary metabolites. Fermentation extracts were evaluated, looking for new antifungal activities against plant and human fungal pathogens, as well as, cytotoxic activities against the human liver cancer cell line HepG2. From the 349 isolates tested, 126 (36%) exhibited signifcant bioactivities including 58 strains with exclusive antifungal properties and 33 strains with exclusive activity against the HepG2 hepatocellular carcinoma cell line. After LCMS analysis, 68 known bioactive secondary metabolites could be identifed as produced by 96 strains, and 12 likely unknown compounds were found in a subset of 14 fungal endophytes. The chemical profles of the diferential expression of induced activities were compared. As proof of concept, ten active secondary metabolites only produced in the presence of resins were purifed and identifed. The structures of three of these compounds were new and herein are elucidated. Several bioprospecting reports have published the ability of endophytic fungi to produce a broad range of bioac- tive secondary metabolites. Many of them are used as sources of anticancer lead compounds (i.e. taxol, vincristine, vinblastine, camptothecin and podophyllotoxin)1 or as sources of antifungal lead molecules (i.e., cryptocandin A2, enfumafungin3, CR3774, ambuic acid5, jesterone6, moriniafungin7, parnafungins8 or phaeofungin9). Fungal endophytes represent one of the most prolifc sources of novel natural products but, low production yields and the lack of expression of cryptic gene clusters in laboratory conditions are frequently key limiting factors to exploit their high potential. Several strategies have been developed to address these issues, including mutagenesis, genetic transformation, agar co-cultivation, mixed-culture fermentations and the use of additives, such as epigenetic modifers or adsorptive polymeric resins10. We recently reported the promotion of new chem- ical entities by the addition of Diaion® and Amberlite® resins to arrays of fungal fermentation media. Results 1Fundación MEDINA, Avda. del conocimiento 34, 18016, Granada, Spain. 2Departamento de Botánica, Facultad de Farmacia, Universidad de Granada, C/ Prof. Clavera, s/n, 18011, Granada, Spain. Correspondence and requests for materials should be addressed to V.G.-M. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:9729 | DOI:10.1038/s41598-018-28192-5 1 www.nature.com/scientificreports/ concluded that this approach can induce the production of new secondary metabolites and afect consistently the production yields, at least for specifc groups of fungal strains11. The Iberian southeast is one of the most arid regions in Europe. It is characterized by a warm and dry Mediterranean climate and it is rich in gypsum and saline soils. Tese unique ecological conditions have allowed the development of a wide range of endemic plant species where it is frequent to fnd specimens of the Aizoaceae, Chenopodiaceae or Plumbaginaceae families12 which are barely represented outside of these environments13. Te diversifcation of Ascomycota, the highest speciated fungal phylum, is refected in its multiple symbiotic strategies with vascular plants, and symbiotic lifestyles (mutualistic, commensalistic, parasitic and pathogenic) in response to host genotype and environmental factors14. Fungal endophytes are mostly known to infect plants without causing symptoms. Latent pathogens seem to represent a relatively small proportion of the endophytic community, including latent saprophytes and mutualistic species15. A partial characterization of Ascomycotina from arid zones of Almeria and Baetic mountains (Andalusia) indicates that fungi associated to plants of these areas remain to be discovered16. Limited information is still available on the biodiversity of fungal symbionts and host-specifc endophytes in these areas, including the role of these microorganisms in the ecological ftness and survival of arid plants. Previous studies on fungal endophytes from plants of arid areas17 have not described their potential to gener- ate chemical diversity, they only provided scattered examples of these populations within specifc medicinal plant hosts18,19 and mentioned their bioactive potential as a whole. With this in mind, we decided to perform a broader survey in Andalusia, one of the richest areas in arid plant endemisms of Europe, isolating and characterizing both, morphologically and chemically these endophytes and their potential to generate new bioactive secondary metabolites by selecting the best combinations of media and resins to promote their chemical diversity generation based on previous results11. Results Biodiversity of fungal isolates. From the individual plant species collected (63) a total of 349 fungal strains were isolated. 310 were obtained from surface-disinfected leaf or stem pieces, 32 were directly isolated from fungal reproductive structures on stems, and 7 were isolated directly from cleistothecia and/or conidi- ophores, which developed on plant material afer incubation in moist chambers. Given the major interest in low occurring fungal species colonizing these substrates, 286 strains (45.0%) from dominant species such as Cladosporium, Penicillium and Aspergillus were not considered in further studies as they are generally considered as ubiquitous epiphytes. The remaining isolates were distributed among 19 orders including Basidiomycetes and Ascomycetes (Supplementary Information Fig. S2). More than half of the isolates (208; 59.6%) were included within the Pleosporales order, followed by Dothideales (22; 6.0%) and Xylariales (19; 5.5%). Within the Pleosporales order, our isolates were distributed among 42 genera belonging to 14 families, being the Pleosporaceae, Didymellaceae and Sporormiaceae the most abundant with 76, 43 and 19 isolates respectively. In total, 107 diferent genera were iden- tifed being the 10 genera with the highest number of isolates: Neocamarosporium (37), Preussia (19), Alternaria (18), Ascochyta (17), Phoma (14), Comoclathris (13), Neomicrosphaeropsis (10), Aureobasidium (7), Pleospora (7) and Fusarium (7) (see full list in Supplementary Information, Table S1). Phylogenetic analyses. Phylogenetic analysis of the isolates belonging to the above-mentioned genera within the Pleosporales order were based on ITS/28S rDNA excluding Preussia which was the subject of a specifc review study recently published by our group20. Te diferent runs of the Bayesian and ML analyses yielded the same topology. Te consensus phylogenetic tree of the 130 isolated strains with 95 GenBank sequences of rep- resentative strains (see Table S2 in Supplementary information) included the endophytic strains™ isolated recently from other plants of the Arizona desert17,21. Te resulting tree also showed a very similar topology to the phyloge- netic trees obtained recently in other Pleosporales characterization studies22–24. Te ITS/28S rDNA tree revealed six main clades (Fig. 1): Te Coniothyriaceae clade clustered Coniothyrium and Hazslinszkyomyces species and included six of our isolates supported statistically (posterior probability val- ues = 91%/maximum likelihood bootstrap = 95%); Te Incertae sedis clade grouped Camarosporium species including the Camarosporium isolate CF-285350 with high statistical support (pp = 100%/bs = 100%); Te highly supported Montagnulacea clade grouped three diferent genera, Pseudocamarosporium (pp = 100%/bs = 98%), Paracamarosporium (pp = 100%/bs = 100%) and Kalmusia (pp = 99%/bs = 97%) including six of our isolates; Te Didymellaceae clade, including species of Xenodidymella, Neodidymelliopsis, Neomicrosphaeropsis, Didymella, Leptosphaerulina and Ascochyta grouped 28 of our isolates with moderate statistical support (pp = 92%/bs = 89%); Te highly supported Leptosphaeriaceae clade (pp = 100%/bs = 100%) included the Neosetophoma species and three Phoma-like